Site-directed Modification And Functional Analysis Of Aspergillus Ochraceus P450 Monooxygenase

Eplerenone is a drug that protects the cardiovascular system.11?-Hydroxycanrenone is the key intermediate in eplerenone synthesis.An Aspergillus ochraceus MF016 which could catalyse the conversion of canrenone to 11?-hydroxycanrenone utilizing cytochrome P450(CYP)enzyme system was isolated in our laboratory.However,its biocatalytic efficiency is low.In addition,the ochratoxin A(OTA)co-catalyzed by two polyketide synthases,a secondary metabolite produced by this strain,has strong liver and kidney toxicity.It also has potential carcinogenic and mutational capabilities.Those disadvantages severely restrict the application of A.ochraceus MF016 in related fields such as pharmaceuticals and foods industry.To date,many studies had focused on the optimisation of the fermentation conditions of steroids produced by A.ochraceus,but few have focused on the genetic modification of A.ochraceus.The genetic modification can resolve the key technical difficulties and improve the efficiency of steroid catalysis.To obtain the recombinant A.ochraceus strain with the high expression of CYP monooxygenase without OTA production,the genome sequence of A.ochraceus MF016(Gen Bank No.VBTP00000000)was determined by Pac Bio sequel systems powered via single-molecule real-time(SMRT)sequencing technology.The two polyketide synthase gene pks1 and pks2 was predicted based on the whole-genome sequencing results.The gene pks2 was selected for knockout.Primers was designed based on pks2 sequence for the cloning of gene fragment pks2-ud.Then,the pks2-ud was inserted into the Escherichia coli-Aspergillus shuttle vector p BC-Hygro to construct the recombinant plasmid p B-pks2-ud.Furthermore,the pks2-ud gene sequence in the plasmid p B-pks2-ud was disrupted via inserting the bleomycin gene with Tr promoter and terminator sequences to construct the recombinant plasmid p B-p1zp2.Protoplasts of A.ochraceus MF016 were prepared.The plasmid p B-p1zp2 was transformed by the PEG-mediated method.A mutant A.ochraceus MF018 with the knockout of pks2 was then obtained by homologous recombination.The mutant strain MF018 lost the ability to produce OTA.Based on the strain MF018,the CYP monooxygenase coding gene of A.ochraceus MF016 was predicted and cloned.Fusion PCR was performed to merge the upstream homology arm sequence of cytochrome P450 monooxygenase gene,promoter sequence,cytochrome P450 monooxygenase gene,hygromycin resistance gene and downstreamhomology arm sequence of cytochrome P450 monooxygenase gene fragments.The constructed complete fusion fragment(named UTPHD)was inserted into the vector p BC-Hygro to construct the recombinant plasmid p B-UTPHD.The A.ochraceus MF018 protoplasts were prepared and the p B-UPTHD was transformed.Then,the recombinant A.ochraceus MF010 with high expression of cytochrome P450 monooxygenase was obtained by homologous recombination.The biocatalytic rate of this recombinant strain MF010 reached approximately 93% at 60 h without adding organic solvents or surfactants,which was17%–18% higher than that of the original strain MF016.Moreover,its biocatalytic time reduced by more than 30 h compared with that of the MF016 strain.These results indicated that the conversion rate of A.ochraceus MF010 is superior to that of A.ochraceus MF016,with reduced biocatalytic time was significantly shortened and lost the ability to produce OTA.During eplerenone synthesis,microbial catalytic conversion from canrenone to11?-hydroxycanrenone is the most critical step.The recombination strain MF010 constructed in this study could improve the biocatalytic efficiency of the 11?-hydroxylation without producing OTA.This strain can overcome the limitation of substrate biocatalytic efficiency and thus holds a high potential for application in the industrial production of eplerenone.